Immunoadjuvant compounds and uses thereof

ABSTRACT

Cyclic beta glucan compounds function as an immunoadjuvant when administered prior to, concommitantly with, or subsequent to the administration of one or more antigens to a subject. These adjuvant compounds may be effectively used as dendritic cell activating molecules.

FIELD OF THE INVENTION

The present invention relates to a cyclic beta glucan compound for useas an immunoadjuvant and vaccine composition comprising thereof.

BACKGROUND OF THE INVENTION

The development of safe and efficacious vaccines remains a major goal inglobal public health.

The majority of the present day vaccines are composed of two maincomponents: (i) the target antigen of therapeutic interest and (ii)immunoadjuvant(s) that stimulate and/or induce immunogenicity againstsaid antigen.

The nature of known immunoadjuvants varies greatly, but includes inparticular mineral oils, bacterial extracts, live and attenuatedorganisms and suspensions of aluminum hydroxide metals.

Even if immunoadjuvants provide enhanced immune responses, their use canalso elicit adverse side effects, function notably of their administeredroute. Therefore, the numbers of immunoadjuvants that are approved andeffective in humans remain relatively limited. Accordingly there is aneed for new compounds which could be used as immunoadjuvants withouttriggering adverse side effects such as endotoxic side effect.

The immune response to bacterial infection relies on the combined actionof both the innate and adaptive immune systems. Dendritic Cells (DCs)are the most efficient professional antigen-presenting cells APCs, whichplay an important role initiation and regulation of immune response. DCsare critical sentinels that detect, capture, and process antigens, suchas invading bacteria and virus, and have the ability to migrate fromperipheral tissues to secondary lymphoid organs to elicit primary T cellresponses. Upon exposure to microbial stimuli, DCs undergo a maturationprocess characterized by the increased formation of MHC-peptidecomplexes, the up-regulation of co-stimulatory molecules (CD86, CD40 andCD80) and the cytokine production. Besides, other hallmarks of DCmaturation process are the induction of chemokine receptors thatfacilitate movement into regional lymph nodes (CCR7) and the increaseability to activate T cells.

DCs recognize microbial stimuli, also called pathogen associatedmolecular patterns (PAMPs), by highly conserved receptorspattern-recognition receptors (PRRs). The best known and characterizedclasses of PRRs are the Toll-like receptors (TLRs) and C-type lectinreceptors (CLRs). For example, lipoproteins and peptidoglycan arerecognized by TLR2, dsRNA by TLR3, LPS by TLR4, CpG by TLR9, flagellinby TLR5, ssRNA by TLR7/8, CpG by TLR9, mannose-containing molecules byDC-SIGN and linear β-glucan by Dectin-1. When these receptors aretriggered, downstream signalling cascades are activated for induction ofinflammatory responses. Signalling pathways activated following TLRengagement can vary, depending on the recruitment or not of MyD88.MyD88-independent pathway that is unique to TLR3 and TLR4 leads to theexpression of interferon regulatory factor 3 while MyD88-dependentsignalling pathway, present on all TLRs except for TLR3, converge onMAPKs and NF-κB induction to exert their biological effects in fine.

Osmoregulated periplasmic glucans (OPGs) are general constituents of theperiplasmic space of Gram-negative bacteria envelopes (22). They havebeen found in all the proteobacteria tested. OPGs exhibit quitedifferent structures among various species but they share several commoncharacteristics: (i) they are oligosaccharides made of a limited numberof units (5 to 24); (ii) D-glucose is the only constituent sugar; (iii)glucose units are linked, at least partially, by β-glycosidic bonds;(iv) glucan concentration in the periplasm increases in response to adecrease of environmental osmolarity. OPGs seem to have a criticalbiological function because mutants deficient in OPG synthesis presenthighly pleiotropic phenotype (eg chemotaxis, motility, reduced outermembrane stability and synthesis of exopolysaccharides as well asdefective growth in hypoosmotic media) (22). Besides, they are unable toestablish successful pathogenic or symbiotic associations witheukaryotic hosts (1).

Brucella is a α-Proteobacteria considered as facultative intracellularpathogens of mammals, including humans. The pathogenesis of theresulting zoonosis, called brucellosis, is mostly linked to the abilityof Brucella to survive and replicate intracellularly, in bothprofessional and non-professional phagocytic host cells. In Brucellaspp. cyclic beta glucans (CβG) consists of a cyclic backbone with adegree of polymerization ranging from 17 to 25, in which all the glucoseunits are linked by β-1,2 linkages (Brucella CβG) (2). It has beendescribed that the presence of cyclic glucan is required for full B.abortus virulence (1). Moreover, Arellano-Reynoso et al. (3) determinedthat Brucella CβG, which modulates lipid microdomain organization, wasessential for preventing lysosome fusion and allowing Brucella to reachits replicative niche. CβG are expressed in large amounts, representing1-5% of the bacteria dry weight (ref). Therefore, considering that ifthe content of a single bacterium is released inside aBrucella-containing vacuole, the volume of which is about 10femptoliter, the concentration of the CβG in the vacuole would be of amM range. This means that when thousands of bacteria released fromapoptotic cells die, CβG released in the external medium can beestimated in the μM range and this may have some important consequenceson the immune system.

The role of glucans and especially linear beta glucans as importantPAMPs involved in host-pathogen interactions (4, 5) has been extensivelydescribed. Interestingly, linear (1 - - - 3) β-glucans are recognizedfor their immunomodulatory properties, because they have been shown topossess antitumor (6) and anti-infective properties against bacterial(7), viral (8), fungal (9), and protozoal (10) infections. However, todate, there is no report about the properties of cyclic β-glucans asmodulators of the immune system.

SUMMARY OF THE INVENTION

The inventors have been demonstrated that Brucella cyclic beta glucans(CβG) exhibit an immunoadjuvant activity as illustrated in the examplesherein.

It has been also shown according to the present invention that thesenovel adjuvant compounds represent in particular a new class ofdendritic cell activating molecules. The inventors for the first timedemonstrate that Brucella CβG is a TLR4 agonist and a potent activatorof mouse and human DCs since it is capable of inducing dendritic cellmaturation, pro-inflammatory cytokine secretion and activation andproliferation of both CD4 and CD8 T cells.

Said new cyclic beta glucan compounds of the invention are thusparticularly useful as immunoadjuvants, to induce and/or to enhance animmune response.

The present invention thus relates to an immunoadjuvant compoundcomprising at least one cyclic beta glucan compound.

The invention also relates to a vaccine composition comprising animmunoadjuvant compound as defined above, one or more antigens, andoptionally one or more pharmaceutically acceptable excipients.

The present invention also relates to the immunoadjuvant compound asdefined above, for use as a medicament (in particular to induce and/orto enhance adjuvant activity).

This invention also concerns the use of an immunoadjuvant compoundaccording to the invention, for manufacturing a vaccine composition, inparticular for inducing and/or for enhancing an immune response againstone or more antigens.

The invention also relates to a kit containing:

-   -   an immunoadjuvant compound according to the invention,    -   at least one antigen;

as combined preparation for simultaneous, separate or sequential use toinduce a protective immune response against, for example, a pathogen, orto efficaciously protect the subject or the animal against infection.

A further object of the invention relates to a cyclic beta glucancompound for use in a therapeutic method for inducing maturation ofdendritic cells (DCs) in a subject in need thereof.

Another further object of the invention relates to a cyclic beta glucancompound for use as immunoadjuvant, wherein said compound is use forinducing maturation of dendritic cells (DCs) in a subject in needthereof.

DETAILED DESCRIPTION OF THE INVENTION

The inventors have investigated the effect of Brucella cyclic betaglucans (CβG) and other cyclic glucans on DCs maturation, in term ofproduction of cytokines, surface expression of MHC-II and co-stimulatorymolecules, gene expression, toxicity and antibody responses on bothhuman and mouse DCs. They have demonstrated that Brucella CβG are potentactivators of mouse and human DCs. Furthermore, cyclic glucans withdifferent structures did not induce the same activation responses,showing that CβG with different structures may have different activationproperties. The inventors have also demonstrated that CβG represents anovel class of TLR4 agonists. It was indeed demonstrated that saidcyclic beta glucan has a TLR4-mediated adjuvant properties, with in vivopro-inflammatory effect, but do not shows any significant systemicendotoxic side effect after systemic injection. The results contained inthe examples herein show that the said cyclic beta glucan does not showsignificant intrinsic antigenic effect, i.e. the molecule of interestprevents or attenuates the potency to trigger cyclic beta glucanantibodies, notably into serum. Accordingly, the inventors demonstratethat CβG could represent a next class of immunoadjuvant.

Accordingly, a first aspect of the invention relates to animmunoadjuvant compound comprising at least one cyclic beta glucan (CβG)compound.

The present invention also relates to a cyclic beta glucan compound foruse as an immunoadjuvant.

As used herein, the term “immunoadjuvant” refers to a compound that caninduce and/or enhance the immune response against an antigen whenadministered to a subject or an animal. It is also intended to mean asubstance that acts generally to accelerate, prolong, or enhance thequality of specific immune responses to a specific antigen. In thecontext of the present invention, the term “immunoadjuvant” means acompound, which enhances both innate immune response by affecting thetransient reaction of the innate immune response and the more long-livedeffects of the adaptive immune response by activation and maturation ofthe antigen-presenting cells (APCs) especially Dentritic cells (DCs).

Accordingly a further object of the invention relates to a cyclic betaglucan compound for use in a therapeutic method for inducing maturationof dendritic cells (DCs) in a subject in need thereof.

Another further object of the invention relates to a cyclic beta glucancompound for use as immunoadjuvant, wherein said compound is use forinducing maturation of dendritic cells (DCs) in a subject in needthereof.

As used herein the term “antigen” refers to a molecule capable of beingspecifically bound by an antibody or by a T cell receptor (TCR) ifprocessed and presented by MHC molecules. The term “antigen”, as usedherein, also encompasses T-cell epitopes. An antigen is additionallycapable of being recognized by the immune system and/or being capable ofinducing a humoral immune response and/or cellular immune responseleading to the activation of B- and/or T-lymphocytes. An antigen canhave one or more epitopes or antigenic sites (B- and T-epitopes).

As used herein, the term “cyclic beta glucan compound” or “CβG compound”refers to a low-molecular-weight cell surface carbohydrate with a cyclicbackbone comprising between 17 and 25 hexose residues linked solely bybeta-(1,2) glycosidic bonds. The hexose residues may be selected fromthe group consisting of naturally occurring hexose units and as well asstructural alterations which incorporate non-naturally occurringhexoses, hexose analogs and mimetics. Those skills in the art know orcan determine what structure constitutes functionally equivalent hexoseanalogs and hexose mimetics. Preferably, the naturally occurring hexoseunits may be selected from the group consisting of aldohexose which canform cyclic structure like allose, altrose, galactose, glucose, gulose,idose, mannose and talose. Preferably, naturally occurring hexose unitsare glucose.

In some embodiment various molecules can be attached to the cyclic betaglucan compound of the invention, covalently by using the free hydroxylegroups located on hexose units and/or non covalently by chargeinteractions created by the shape of the doughnut-like ring displaying aniche of about 10 A accepting molecules (11). The molecules may beselected from the group consisting of polypeptides, carbohydrates,nucleic acids or lipids (like cholesterol).

In some embodiments, the hexose residues may be substituted with atleast one natural substituent residue. The “natural substituent residue”could means succinyl (Suc) residues (like in Brucella abortus CβG andSinorhizobium melitoti CβG), phosphoglycerol residues (P-Gro) (like inE. coli CβG and Sinorhizobium melitoti CβG); phosphoethanolamine (P-Etn)residues (like in E. coli CβG);, phosphocholine residues (P-Cho) (likein Bradyrhizobium japonicum CβG);, acetyl (Ace) residues (like in R.sphaeroides CβG); methylmalonyl (MeMal) residues (like in S. melilotiCPG). In preferred embodiments, the natural substituent residue issuccinyl residues. Such substitution occurs naturally in cyclic betaglucan of other bacterial species like Brucella abortus (O-succinylresidues). Other substitutions can be introduced in positions that donot alter the immunoadjuvant activity of the CβG. Typically, thesubstituent linkage on cyclic beta glucan compound is preferably made upof O-ester link on hydroxyl groups of the hexose. In some embodimentsthe number of natural substituent residue is comprise between, 0 to 25.In preferred embodiments the number of natural substituent residue iscomprise between 0 to 3.

The skilled man in the art can evaluate easily the CβG compound havingimmunoadjuvant properties according to the invention by testing whethersaid compound induce maturation of dendritic cells by measuring cytokineproduction such as IL-12p and TNF-alpha production and expression ofsurface marker on dentritic cells such as CD80, CD40 and CD86. In secondstep, induction of CD8+T cell proliferation and activation can also betested. Alternatively TLR4 agonistic activities may also be determined.Typically, the tests that may be used to test the immunodjuvant activityof a CβG compound are described in the Example.

The cyclic beta glucan compound of the invention may be obtained bypurification of a bacterium, more preferably from Brucella, or by anyone of the methods for chemical or oligosaccharides synthesis, that arewell known from the one skilled in the art.

For example cyclic beta glucan compounds of interest may be recoveredfrom culture medium or from bacterium cell lysates. Typically, the CβGcompounds of the invention are isolated from Brucella. For example theCβG compound of the invention may be isolated from wild type Brucellacells or from Brucella strains such as Brucella melitensis 16M (AmericanType Culture Collection 23456; virulent strain, biotype 1), Brucellaabortus 2308 (12). Bacterium employed in production of the cyclic betaglucan of interest can be disrupted by various physical or chemicalmeans, such as freeze-thaw cycling, sonication, mechanical disruption,or cell-lysing agents.

It may be desired to purify the cyclic beta glucan of interest frombacteria. The procedures described in the Example are exemplary ofsuitable procedures for purification of CβGs from the supernatant ofBrucella melitensis or Brucella abortus.

The following procedures are exemplary of suitable purificationprocedures of cyclic beta glucan compound: by fractionation on anion-exchange column; ethanol precipitation (as used in the example);reverse phase HPLC; chromatography on silica or on a cation-exchangeresin such as DEAE; chromatofocusing; SDS-PAGE; ammonium sulfateprecipitation; gel filtration using, for example, SEPHADEX G-75; ProteinA SEPHAROSE columns to remove contaminants; and metal chelating columnsto bind epitope-tagged forms of the cyclic beta glucan of interest.Various methods of oligosaccharide purification may be employed and suchmethods like trichloracetic acid treatment and gel permeationchromatography are known in the art and described, for example, in (21).

The purification step(s) selected will depend, for example, on thenature of the production process used and the particular cyclic betaglucan compound produced.

Preparation and production of cyclic beta glucan compound, and speciallywith β-1,2 linkages, by cultivating a bacterium (Agrobacterium orRhizobium), and collecting the titled compound from the culture solutionare also described in JP 61040799 and JP61070994.

In certain embodiments, cyclic beta glucan compound of the invention maybe synthesised through conventional techniques of chemical synthesis.For example international patent application WO 0216384 describesapparatus and methods for the automated synthesis of oligosaccharidesfor the efficient synthesis of oligosaccharides on a solid support,e.g., formed by subunit addition to terminal subunits immobilized onsolid-phase particle. Cyclic beta glucan compound of the invention mayalso be synthesised by using the laminarinase 16A glycosynthase (23).

A further object of the invention relates to a vaccine composition,comprising a CβG compound according to the invention as animmunoadjuvant optionally with one or more pharmaceutically acceptableexcipients. More particularly, the present invention pertains to avaccine composition comprising an immunoadjuvant compound as definedabove, together with one or more antigens.

A “vaccine composition”, once it has been administered to a subject oran animal, elicits a protective immune response against said one or moreantigen(s) which is (are) comprised herein. Accordingly, the vaccinecomposition of the invention, once it has been administered to thesubject or the animal, induces a protective immune response against, forexample, a microorganism, or to efficaciously protect the subject or theanimal against infection.

A variety of substances can be used as antigens in a compound orformulation, of immunogenic or vaccine type. For example, attenuated andinactivated viral and bacterial pathogens, purified macromolecules,polysaccharides, toxoids, recombinant antigens, organisms containing aforeign gene from a pathogen, synthetic peptides, polynucleic acids,antibodies and tumor cells can be used to prepare (i) an immunogeniccomposition useful to induce an immune response in a individual or (ii)a vaccine useful for treating a pathological condition.

Therefore, a cyclic beta glucan compound of the invention can becombined with a wide variety of antigens to produce a vaccinecomposition useful for inducing an immune response in an individual.

Those skilled in the art will be able to select an antigen appropriatefor treating a particular pathological condition and will know how todetermine whether an isolated antigen is favored in a particular vaccineformulation.

Those skilled in the art will be also able to determine whether it ispreferable to covalently link, or not covalently link, theimmunoadjuvant of the invention to the said one or more antigens. Thus,in a further aspect of the invention, the present invention relates tocyclic beta glucan compound according to the invention linked to atleast one antigen.

An isolated antigen can be prepared using a variety of methods wellknown in the art. A gene encoding any immunogenic polypeptide can beisolated and cloned, for example, in bacterial, yeast, insect, reptileor mammalian cells using recombinant methods well known in the art anddescribed, for example in Sambrook et al., Molecular cloning : ALaboratory Manual, Cold Spring Harbor Laboratory, New York (1992) and inAnsubel et al., Current Protocols in Molecular Biology, John Wiley andSons, Baltimore, Md. (1998). A number of genes encoding surface antigensfrom viral, bacterial and protozoan pathogens have been successfullycloned, expressed and used as antigens for vaccine development. Forexample, the major surface antigen of hepatitis B virus, HbsAg, the Psubunit of choleratoxin, the enterotoxin of E. coli, thecircumsporozoite protein of the malaria parasite, and a glycoproteinmembrane antigen from Epstein-Barr virus, as well as tumor cellantigens, have been expressed in various well known vector/host systems,purified and used in vaccines.

A pathologically aberrant cell may also be used in a vaccine compositionaccording to the invention can be obtained from any source such as oneor more individuals having a pathological condition or ex vivo or invitro cultured cells obtained from one or more such individuals,including a specific individual to be treated with the resultingvaccine.

In a particular embodiment, the antigen of the vaccine composition couldbe a “Tumor associated antigen”. As used herein, the term “tumorassociated antigen” refers to an antigen that is characteristic of atumor tissue. An example of a tumor associated antigen expressed by atumor tissue may be the antigen prostatic acid phosphatise (see WO2004026238) or MART peptide T (melanoma antigen).

The vaccine composition according to the invention may contain at leastone other immunoadjuvant. A variety of immunoadjuvant may be suitable toalter an immune response in an individual. The type of alterationdesired will determine the type of immunoadjuvant selected to becombined with the said cyclic beta glucan compound of the invention. Forexample, to enhance the innate immune response, the vaccine compositionof the invention can comprise another immunoadjuvant that promotes aninnate immune response, such as other PAMP or conserved region known orsuspected of inducing an innate immune response. A variety of PAMPs areknown to stimulate the activities of different members of the toll-likefamily of receptors. Such PAMPs can be combined to stimulate aparticular combination of toll-like receptors that induce a beneficialcytokine profile. For example, PAMPs can be combined to stimulate acytokine profile that induces a Th1 or Th2 immune response. Other typesof immunoadjuvant that promote humoral or cell-mediated immune responsescan be combined with a cyclic beta glucan compound of the invention. Forexample, cytokines can be administered to alter the balance of Th1 andTh2 immune responses. Those skilled in the art will know how todetermine the appropriate cytokines useful for obtaining a beneficialalteration in immune response for a particular pathological condition.

In another particular embodiment, the vaccine composition according tothe invention, further comprises one or more components selected fromthe group consisting of surfactants, absorption promoters, waterabsorbing polymers, substances which inhibit enzymatic degradation,alcohols, organic solvents, oils, pH controlling agents, preservatives,osmotic pressure controlling agents, propellants, water and mixturethereof.

The vaccine composition according to the invention can further comprisea pharmaceutically acceptable carrier. The amount of the carrier willdepend upon the amounts selected for the other ingredients, the desiredconcentration of the antigen, the selection of the administration route,oral or parenteral, etc. The carrier can be added to the vaccine at anyconvenient time. In the case of a lyophilised vaccine, the carrier can,for example, be added immediately prior to administration.Alternatively, the final product can be manufactured with the carrier.

Examples of appropriate carriers include, but are not limited to,sterile water, saline, buffers, phosphate-buffered saline, bufferedsodium chloride, vegetable oils, Minimum Essential Medium (MEM), MEMwith HEPES buffer, etc.

Optionally, the vaccine composition of the invention may containconventional, secondary adjuvants in varying amounts depending on theadjuvant and the desired result. The customary amount ranges from about0.02% to about 20% by weight, depending upon the other ingredients anddesired effect. For the purpose of this invention, these adjuvants areidentified herein as “secondary” merely to contrast with theabove-described immunoadjuvant compound that is an essential ingredientin the vaccine composition for its effect in combination with anantigenic substance to significantly increase the humoral immuneresponse to the antigenic substance. The secondary adjuvants areprimarily included in the vaccine formulation as processing aidsalthough certain adjuvants do possess immunologically enhancingproperties to some extent and have a dual purpose.

Examples of suitable secondary adjuvants include, but are not limitedto, stabilizers; emulsifiers; aluminum hydroxide; aluminum phosphate; pHadjusters such as sodium hydroxide, hydrochloric acid, etc.; surfactantssuch as Tween® 80 (polysorbate 80, commercially available from SigmaChemical Co., St. Louis, Mo.); liposomes; iscom adjuvant; syntheticglycopeptides such as muramyl dipeptides; extenders such as dextran ordextran combinations, for example, with aluminum phosphate;carboxypolymethylene; bacterial cell walls such as mycobacterial cellwall extract; their derivatives such as Corynebacterium parvum;Propionibacterium acne; Mycobacterium bovis, for example, BovineCalmette Guerin (BCG); vaccinia or animal poxvirus proteins; subviralparticle adjuvants such as orbivirus; cholera toxin;N,N-dioctadecyl-N′,N′-bis(2-hydroxyethyl)-propanediamine (pyridine);monophosphoryl lipid A; dimethyldioctadecylammonium bromide (DDA,commercially available from Kodak, Rochester, N.Y.); synthetics andmixtures thereof. Desirably, aluminum hydroxide is admixed with othersecondary adjuvants or an immunoadjuvant such as Quil A.

Examples of suitable stabilizers include, but are not limited to,sucrose, gelatin, peptone, digested protein extracts such as NZ-Amine orNZ-Amine AS. Examples of emulsifiers include, but are not limited to,mineral oil, vegetable oil, peanut oil and other standard,metabolizable, nontoxic oils useful for injectables or intranasalvaccines compositions.

Conventional preservatives can be added to the vaccine composition ineffective amounts ranging from about 0.0001% to about 0.1% by weight.Depending on the preservative employed in the formulation, amounts belowor above this range may be useful. Typical preservatives include, forexample, potassium sorbate, sodium metabisulfite, phenol, methylparaben, propyl paraben, thimerosal, etc.

The vaccine composition of the invention can be formulated as a solutionor suspension together with a pharmaceutically acceptable medium.

Such a pharmaceutically acceptable medium can be, for example, water,phosphate buffered saline, normal saline or other physiologicallybuffered saline, or other solvent or vehicle such as glycol, glycerol,and oil such as olive oil or an injectable organic ester. Apharmaceutically acceptable medium can also contain liposomes ormicelles, and can contain immunostimulating complexes prepared by mixingpolypeptide or peptide antigens with detergent and a glycoside, such asQuil A.

Liquid dosage forms for oral administration of the vaccine compositionof the invention include pharmaceutically-acceptable emulsions,microemulsions, solutions, suspensions, syrups and elixirs. In additionto the active ingredient(s), the liquid dosage forms may contain inertdiluents commonly used in the art, such as, for example, water or othersolvents, solubilizing agents and emulsifiers, such as ethyl alcohol,isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol,benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (inparticular, cottonseed, groundnut, corn, germ, olive, castor and sesameoils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fattyacid esters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active ingredient(s), may containsuspending agents as, for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth,and mixtures thereof.

Formulations of the vaccine compositions of the invention for rectal orvaginal administration may be presented as a suppository, which may beprepared by mixing the active ingredient(s) with one or more suitablenon-irritating excipients or carriers comprising, for example, cocoabutter, polyethylene glycol, a suppository wax or salicylate and whichis solid at room temperature, but liquid at body temperature and,therefore, will melt in the rectum or vaginal cavity and release theactive ingredient(s). Formulations of the present invention which aresuitable for vaginal administration also include pessaries, tampons,creams, gels, pastes, foams or spray formulations containing suchcarriers as are known in the art to be appropriate

Vaccine compositions of this invention suitable for parenteraladministration comprise the active ingredient(s) in combination with oneor more pharmaceutically-acceptable sterile isotonic aqueous ornon-aqueous solutions, dispersions, suspensions or emulsions, or sterilepowders which may be reconstituted into sterile injectable solutions ordispersions just prior to use, which may contain antioxidants, buffers,solutes which render the formulation isotonic with the blood of theintended recipient or suspending or thickening agents.

Examples of suitable aqueous and non-aqueous carriers which may beemployed in the vaccine compositions of the invention include water,ethanol, polyols (such as glycerol, propylene glycol, polyethyleneglycol, and the like), and suitable mixtures thereof, vegetable oils,such as olive oil, and injectable organic esters, such as ethyl oleate.Proper fluidity can be maintained, for example, by the use of coatingmaterials, such as lecithin, by the maintenance of the required particlesize in the case of dispersions, and by the use of surfactants.

These compositions may also contain adjuvants such as wetting agents,emulsifying agents and dispersing agents. It may also be desirable toinclude isotonic agents, such as sugars, sodium chloride, and the likein the compositions. In addition, prolonged absorption of the injectablepharmaceutical form may be brought about by the inclusion of agentswhich delay absorption such as aluminum monostearate and gelatin.

Injectable depot forms are made by forming microencapsule matrices ofthe active ingredient(s) in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of the activeingredient(s) to polymer, and the nature of the particular polymeremployed, the rate of release of the active ingredient(s) can becontrolled. Examples of other biodegradable polymers includepoly(orthoesters) and poly(anhydrides). Depot injectable formulationsare also prepared by entrapping the active ingredient(s) in liposomes ormicroemulsions which are compatible with body tissue. The injectablematerials can be sterilized for example, by filtration through abacterial-retaining filter.

The formulations may be presented in unit-dose or multi-dose sealedcontainers, for example, ampoules and vials, and may be stored in alyophilized condition requiring only the addition of the sterile liquidcarrier, for example water for injection, immediately prior to use.Extemporaneous injection solutions and suspensions may be prepared fromsterile powders, granules and tablets of the type described above.

The amount of antigen and immunoadjuvant compound in the vaccinecomposition according to the invention are determined by techniques wellknown to those skilled in the pharmaceutical art, taking intoconsideration such factors as the particular antigen, the age, sex,weight, species, and condition of the particular animal or patient, andthe route of administration.

While the dosage of the vaccine composition depends notably upon theantigen, species of the host vaccinated or to be vaccinated, etc., thedosage of a pharmacologically effective amount of the vaccinecomposition will usually range from about 0.01 μg to about 500 μg (andin particular 50 μg to about 500 μg) of the immunoadjuvant compound ofthe invention per dose.

Although the amount of the particular antigenic substance in thecombination will influence the amount of the immunoadjuvant compoundaccording to the invention, necessary to improve the immune response, itis contemplated that the practitioner can easily adjust the effectivedosage amount of the immunoadjuvant compound through routine tests tomeet the particular circumstances.

The vaccine composition according to the invention can be tested in avariety of preclinical toxicological and safety studies well known inthe art.

For example, such a vaccine composition can be evaluated in an animalmodel in which the antigen has been found to be immunogenic and that canbe reproducibly immunized by the same route proposed for human clinicaltesting.

For example, the vaccine composition according to the invention can betested, for example, by an approach set forth by the Center forBiologics Evaluation and Research/Food and Drug Administration andNational Institute of Allergy and Infectious Diseases (13).

Those skilled in the art will know how to determine for a particularvaccine composition, the appropriate antigen payload, route ofimmunization, volume of dose, purity of antigen, and vaccination regimenuseful to treat a particular pathological condition in a particularanimal species.

In a vaccination protocol, the vaccine may be advantageouslyadministered as a unique dose or preferably, several times e.g., twice,three or four times at week or month intervals, according to aprime/boost mode. The appropriate dosage depends upon variousparameters.

As a general rule, the vaccine composition of the present invention isconveniently administered orally, parenterally (subcutaneously,intramuscularly, intravenously, intradermally or intraperitoneally),intrabuccally, intranasally, or transdermally, intralymphatically,intratumorally, intravesically, intraperitoneally and intracerebrally.The route of administration contemplated by the present invention willdepend upon the antigen.

The present invention relates to a kit comprising an immunoadjuvantcompound as defined above (i.e. a CβG compound according to theinvention) and at least one antigen.

More particularly, the invention relates to a kit comprising:

-   -   an immunoadjuvant compound as defined above,    -   at least one antigen as defined above;

as combined preparation for simultaneous, separate or sequential use toinduce a protective immune response against, for example, a pathogen, orto efficaciously protect the subject or the animal against infection.

A CβG compound can be administered prior to, concomitantly with, orsubsequent to the administration of at least one antigen to a subject toinduce a protective immune response against, for example, a pathogen, orto efficaciously protect the subject or the animal against infection.The CβG compound and the antigen are administered to a subject in asequence and within a time interval such that the CβG compound can acttogether with the antigen to provide an increased immune responseagainst said antigen than if they were administered otherwise.Preferably, the CβG compound and antigen are administered simultaneouslyto the subject. Also preferably, the molecules are administeredsimultaneously and every day to said patient.

A further aspect of the invention relates to a method for vaccinating asubject in need thereof comprising administering a pharmacologicallyeffective amount of an antigen and a pharmacologically effective amountof an immunoadjuvant compound according to the invention

As used herein, the term “subject” denotes a mammal, such as a rodent, afeline, a canine, and a primate. Preferably a subject according to theinvention is a human.

A pharmacologically effective amount of the immunoadjuvant compoundaccording to the invention may be given, for example orally,parenterally or otherwise, concurrently with, sequentially to or shortlyafter the administration of the antigen in order to potentiate,accelerate or extend the immunogenicity of the antigen.

The dosage of the vaccine composition will be dependent notably upon theselected antigen, the route of administration, species and otherstandard factors. It is contemplated that a person of ordinary skill inthe art can easily and readily titrate the appropriate dosage for animmunogenic response for each antigen to achieve the effectiveimmunizing amount and method of administration.

The present invention also relates to a TLR4 agonist, which consists ofa CβG compound according to the invention.

As used herein, a TLR4 agonist refers to an agent that is capable ofsubstantially inducing, promoting or enhancing TLR4 biological activityor TLR4 receptor activation or signalling.

A further object of the invention relates to a method for inducing thematuration of dentitic cells (DCs) in a subject in need thereofcomprising administering a pharmacologically effective amount of animmunoadjuvant compound according to the invention.

The invention will be further illustrated by the following figures andexamples. However, these examples and figures should not be interpretedin any way as limiting the scope of the present invention.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B: Induction of mDC maturation depends on the structure ofthe cyclic glucan. Mouse DCs were stimulated for 8 h and 24 h with media(neg), E. coli LPS or B. melitensis C.βG, B. abortus CβG, Ralstonia CβGand MβCD at an equivalent concentration of 0.25 mM. Surface levels ofMHC-II, CD80, CD40 and CD86 were measured by flow cytometry and IL-12and TNF-alpha levels in culture supernatant were determined by ELISA(B). Data are representative of at least three independent experiments.*p<0.05 compared to negative.

FIGS. 2A and 2B. B. melitensis CβG human DCs enhances CD4+ and CD8+ Tcell responses.

A) Human monocyte-derived DCs were stimulated 24 h with media (neg), 10ng/ml of E. coli LPS and 0.2 μM, 2 μM and 20 μM of B. melitensis CβG andHLA-DR, HLA-ABC, CD80, CD40, CD83 and CD86 surface levels were analyzedby FACS. B) Blood mDCs were stimulated with 20 mg/ml C13G for 24 h.Cytokines in the culture supernatants were measured by Luminex.

FIGS. 3A, 3B, and 3C. IL-12 production and CD80, CD40 and CD86 surfaceexpression induced by B. melitensis CβG are dependent on TLR4.

DCs from WT (A, B and C), MyD88KO (A), TRIFKO (A), TLR2KO (B) and TLR4KO(B and C) mice were stimulated for 24 h with media (neg), CpG, PolyI:C,Pam2CSK4 and B. melitensis CβG. A and B) IL-12 concentration in culturesupernatants was determined by ELISA. C) Surface levels of CD80, CD40and CD86 were measured by flow cytometry. As positive controls: Pam2CSK4(100 ng/ml), LPS (100 ng/ml), CpG (1 μM) and Poly I:C (1 mg/ml). Dataare representative of three independent experiments. *p<0.05, comparedto WT mice.

FIGS. 4A and 4B. Adjuvant activity of B. abortus CβG against venom ofBothrops asper.

A; The (**) in the mixture venom+calcium alginate+C13G indicate a valueof p<0.001 with respect to the venom injected alone in a mixture ofcalcium alginate.

B. Adjuvant activity of C13G against the venom of Bothrops asper 21 daysafter immunization.Serum pools of 10 mice per group (total 30 mice). SDis less than 5%.

EXAMPLE 1 Bacterial β1,2 Cyclic Glucans Are Novel Dendritic CellActivators Materials And Methods

Antibodies And Reagents.

Fluorchrome-conjugated antibodies against CD8, CD45.2, CD44, CD25, andCD62L were purchased from BD Biosciences or eBiosciences.

Ovalbumine (OVA) was purchased from EndoGrade with purity>98% andendotoxin concentration <1 EU/mg.

Carboxyfluorescein diacetate succinimidyl ester (CFSE) was ordered fromInvitrogen.

The Aqua Dead Cell Stain for 405 n excitation (Invitrogen) was used todetect dead cells for flow cytometry experiments.

SIINFEKL (SEQ ID NO 1) peptide was purchased from Schafer-N.

Cyclic Glucan Extraction, Purification And Characterization

Cyclic glucan molecules were obtained from B. melitensis 16M (AmericanType Culture Collection 23456; virulent strain, biotype 1), Brucellaabortus 2308 (12) and Ralstonia solanacearum (purified CβG fromRalstonia solanacearum is a gift from Dr. J P Bohin, CNRS UMR8576,Lille, France). The strains were grown in a fermentor at 36° C. and 35%O2 saturation in a biosafety level 3 room in case of Brucella strains(14) and were inactivated with phenol (0.5% at 36° C. for 48 h).Bacteria were resuspended in distilled water (20 g wet weight in 100 ml)and were heated at 120° C. for 30 min. Cell debris was removed (8,000 gfor 30 min at 4° C.) and the extract was precipitated with three volumesof ethanol at −20° C. overnight. The precipitate was removed (5,000 gfor 10 min) and the supernatant was mixed with two volumes of ethanol.The new precipitate was collected (5,000 g for 10 min), was suspended in175 mM NaCl, 0.05% NaN3 and 0.1 M Tris-HCl, pH 7.0, and was digestedfirst for 18 h at 37° C. with nucleases (50 mg of DNase-II type V andRNase-A (Sigma-Aldrich) per ml) and then for 1 h at 55° C. followed by24 h at 25° C. with proteinase K (50 mg/ml; Merck). The fluid wasextracted for 30 min at 70° C. with a volume of phenol, then the mixturewas chilled and centrifuged (8,000 g for 15 min at 0° C.) and theaqueous phase was collected and re-extracted again with phenol using thesame conditions. The waterphase was dialyzed, clarified by briefcentrifugation and freeze-dried. The identity of cyclic glucan wasconfirmed by 13C-nuclear magnetic resonance spectroscopy,high-performance liquid chromatography and high-performance thin layerchromatography (TLC) as described by Aragon and co-workers. The absenceof polysaccharides and lipopolysaccharides or other contaminants wasdemonstrated by ultraviolet spectrophotometry, SDS-PAGE, gelimmunoprecipitation and 3-deoxy-D-manno-2-octulosonic acid determination(14).

Mice And Cells

HEK 293 cells were maintained in DMEM supplemented with 10% FCS.

Wild-type C57BL/6 mice and TLR2−/−, TLR4−/−, MyD88−/−-, TRIF−/− knockout(KO) mice were maintained at Centre d'Immunologie Marseille-Luminy(CIML), Marseille, France. Double (MyD88/TRIF−/−) KO cells were providedby Caetano Reis y Souza laboratory, London, UK.

Mouse bone marrow-derived DCs were prepared from 7-8 week-old femaleC57BL/6 mice or TLR2−/−, TLR4−/−, MyD88−/−, TRIF−/− knockout (KO) miceas previously described (15). Briefly, femurs and tibiae of mice wereremoved and freed of muscles and tendons. The bones were placed in 70%ethanol for 2 min and subsequently washed in PBS. Both bone ends werecut off, and the marrow was flushed out with RPMI 1640 medium. The cellswere seeded in 24 well plates with complete RPMI 1640 (5% FCS and 50 μMβ-mercaptoethanol) supplemented with mouse GM-CSF (mGM-CSF). On day 3,the media was change and on day 6 the experiment was carried out.

Mouse bone marrow-derived macrophages were prepared in the same way asbone marrow-derived DCs, from femurs of 7-8 week-old female C57BL/6mice. Cells were seeds in complete DMEM (2 Mm L-glutamine, 10% FCS)supplemented with M-CSF. On day 5 and 6, media was changed and on day 7the experiment was carried out.

Human monocyte-derived DCs were generated from FICOLL separatedperipheral blood mononuclear cells (PBMCs) from healthy volunteers (16).Monocytes were enriched by adherence and were cultured in complete RPMImedium (supplemented with granulocyte-monocyte colony-stimulating factor(GM-CSF) and IL-4 during 6 days or GM-CSF and IFN during 3 days. Bloodmyeloid DCs (mDCs: HLA−DR+CD11c+CD123−Lin−) were sorted from PBMC usingFACS aria (BD Biosciences, CA). Naive CD4+ and CD8+ T cells(CD45RA+CD45RO−) (purity>99.2%) were purified by FACS-sorting.

C57B1/6 Ly5.1 mice from Jackson Laboratory and OT-I TCR transgenic Ly5.2mice on C57B1/6 background bred in CIML animal facilities were used forexperiments.

Immunofluorescence Microscopy And Flow Cytometry

For immunofluorescence microscopy, stimulated DCs were fixed in 3%paraformaldehyde at 37° C. for 15 min and processed forimmunofluorescence labelling as previously described (17).

Rabbit rivoli antibody against murine I-A (18) and mouse antibody FK2(Biomol) were used as a primary antibodies. After staining, samples wereeither examined on a Leica DMRBE epifluorescencemicroscope or a ZeissLSM 510 laser scanning confocal microscope for image acquisition. Imagesof 102431024 pixels were then assembled using Adobe Photoshop 7.0.Quantification was always done by counting at least 100 cells in 4independent experiments, for a total of at least 400 host cellsanalysed.

For flow cytometry, stimulated DCs were collected and stained.FITC-conjugated antibody to CD80 and CD40, PE-conjugated antibody toCD83 and IA-IE (MHC class II) and APC-conjugated antibody to CD11c wereobtained from Pharmingen. Appropiated isotype antibodies were used ascontrols. After staining, cells were washed with PBS and fixed in 3% ofparaformaldehyde before analysis on a FACScalibur cytometer (BectonDickinson). Cells were always gated on CD11 c for analysis and 10,000gated events were collected from each sample. The data was analysedusing FlowJo software. Histograms were draw from and median fluorescenceintensity values were determinated on gated population. To followproliferation and activation of CFSE-labeled T cells, draining popliteallymph nodes (DLNs) were collected 3 days after the immunization andsubjected to collagenase type I digestion. Cells were stained foranalysis by flow cytometry using different fluorchrome-conjugatedantibodies. At least 100.000 events were collected on FACSCanto II(BDBiosciences). Flow cytometry analysis was performed using FlowJosoftware.

Measurement of Cytokine Concentration

Total mouse IL-12 and TNF-alpha were quantified in culture supernatantof stimulated DCs by sandwich enzyme-linked immunosorbent assays (ELISA)according to the manufacturer's instruction (Abcys). Human cytokines andchemokines, including IL-1b, TNFa, and IL-12p40, were using the BeadLytecytokine assay kit (Upstate, MA) as per the manufacturer's protocol.

Human CD4+ And CD8+ T Cell Responses

5×103 blood mDCs were co-cultured with CFSE-labeled allogeneic naïveCD4+ T cells (1-2×105). Cell proliferation was tested by measuringCFSE-dilution on day 6. CD8+ T cells for 8-10 days in the presence ofIL-2 (20 units/ml). 5×103 blood mDCs from HLA-A0201+ healthy donors wereloaded with 0.2 m.o.i. (multiplicity of infection) heat-inactivatedinfluenza virus (PR8) for 2 h at 37 oC. Autologous CD8+ T cells(1-2×105) were mixed and cultured for 7 days in the presence of 20units/ml IL-2. Cells were then stained with anti-CD8 antibody andtetramer (HLA-A*0201-Flu M158-66). MART-1-specific CD8+ T cell responseswere measured after co-culturing with mDCs loaded with 10 mM MART-126-35(27L) peptide.

Adoptive Transfer of OT-I T Cells And Immunization.

OT-I transgenic cells that express TCR specific for an H-2Db restrictedCD8+ T cell epitope from OVA were used. Lymph nodes OT-I Ly5.2 mice wereharvested and digested with collagenase type I (Sigma) at 37° C. for 30min. CD8+ T cells were then negatively sorted by using mouse CD8negative isolation kit (Dynal). Routinely, the resultant cellswere >90%. CD8 percentage was determined by flow cytometry. CD8+ T cellswere labeled with 10 μM CFSE at 37° C. for 10 min.

CD8+ Ly5.2 CFSE+ T cells were adoptively transferred intravenously(i.v.) into naive congenic C57B1/6 Ly5.1 recipient mice (200 000cells/mouse). 24 h after OT-I adoptive transfer, recipient mice wereimmunized subcutaneously (s.c.) either with 30 μg OVA alone in endotoxinfree PBS or 30 μg OVA mixed with 200 μg of cyclic glucan or 30 μg OVAmixed with 50 μg poly I/C or 30 μg OVA emulsified with IFA(volume/volume).

T Cell Proliferation And Activation.

Viable cells were always gated on CD8+CD45.2+ population and inventorsanalyzed the decrease of CFSE labeling which correlates with thecellular division. To study the cellular activation level, inventorslooked at the expression of activation markers such as CD25, CD44 andCD62L.

HEK 293 Cells Luciferase Reporter Assay

HEK 293 cell reporter assays were performed as described previously ( )using the indicated plasmids. Mouse TLRs, MD2 and CD14 cDNA wereamplified by reverse transcriptase PCR from total RNA prepared from bonemarrow-derived macrophages and subcloning in the pCDNA3.1 expressionvector (Promega). HEK 293 cells were transiently tranfected using Fugene(Roche), according to manufacturer's instructions, for a total of 0.4 μgof DNA consisting of 50 ng of receptor plasmids, 200 ng of pBIIXLucreporter plasmid, 5 ng of control Renilla luciferase (pRL-null,Promega). After 24 h of transfection, cells were stimulated withdescribed agonist for 6 h and then cells were lysed and luciferaseactivity measured using Dual-Glo Luciferase Assay System (Promega).

Results

Brucella CβG Is A Modulator of Mouse DC Maturation

Maturation of mouse DCs is characterized by many morphofunctionalchanges among them, the up-regulation of co-stimulatory and MHC class IImolecules at the cell surface, change in morphology and the formation oflarge cytosolic dendritic cell aggresome-like induced structures, calledDALIS, which are made of defective newly synthesised ubiquitinatedproteins. To initially determinate whether B. melitensis CβG was anactivator of the maturation of mouse DCs, cells were incubated withdifferent concentration of B. melitensis CβG. After 8 and 24 h, surfaceexpression MHC class II molecules and formation of DALIS were analyzedby confocal microscopy. As a control of DC activation, 0.25 mM of E.coli LPS were used. Mouse DCs treated with B. melitensis CβG or E. coliLPS, but not Brucella LPS underwent maturation, since they displayed MHCII surface localization and DALIS formation. However, in untreated DCsand DCs treated with Brucella LPS, MHC II molecules remained mostlyintracellular and DALIS were not observed. the percentage of DCs withDALIS after incubation 8 h and 24 h with the respective stimuli. In thecase of E. coli LPS, 80% of cells contained large and numerous DALISafter 8 h whereas only 20% of non-treated cells contained DALIS. B.melitensis CβG, at 0.025 μM already induced the formation of DALIS in45% of the cells and at 0.25 μM and 2.5 μM the number of cells increasedto 79% and 72%, respectively, reaching the levels obtained with 0.25 μME. coli LPS. At 24 h, the number of DALIS started to decrease, which isconsistent to what has been previously observed since DALIS expressionin the process of DC maturation is transient event.

To obtain further insight into the effect of B. melitensis CβG in the DCmaturation process, inventors next analyzed the surface expression ofclassical maturation markers (CD80, CD86, CD40 and MHC class IImolecules) by flow cytometry in CD11c-positive mouse DCs. Analysis ofthe median of fluorescence showed a clear dose dependent induction ofall co-stimulatory and MHC class II molecules by B. melitensis CβG. Thisis in agreement with microscopy observations where a high proportion ofMHC class II molecules were present on cell surface.

These observations suggest that B. melitensis CβG promotes DCmaturation. Inventors then investigated whether the secretion ofcytokines by mouse DCs was also induced following B. melitensis CβGstimulation. The levels of IL-12 and TNF-alpha in the supernatants ofstimulated mouse DCs were determined. Results showed that B. melitensisCβG induced the secretion of both cytokines in a dose dependent manner.This effect was observed at 8 h as well as at 24 h after stimulation,with no significant differences between both time points. Together,these data confirmed that B. melitensis CβG is a potent activatingmolecule of mouse DCs.

Mouse DC Maturation Is Modulated By Different Cyclic Glucan Structures

Inventors then investigated the possible relationship between cyclicglucan structure and DC activation by incubating DCs with various CβGfrom different origins. B. melitensis CβG consists of a cyclic backbonecontaining 17-25 glucose residues that are linked by β-1, 2 linkages(22). In the case of B. abortus CβG a fraction of the cyclic β-1,2-glucans is substituted with O-succinyl residues (19). Ralstonia C13Gis composed by a cyclic backbone with 13 glucoses. One linkage is α-1, 6whereas all the other glucose residues are linked by β-1, 2 (22). Thesynthetic methyl-cyclodextrin (MβCD) consists of a 7 glucose cyclicblackbone that are linked by β-1, 4-linkages and with O-methylsubstitutions, which is also known for biologists as a lipid raftdestructive agent using its property to extract membrane cholesterol(22).

First inventors analyzed the surface expression of maturation markers.E. coli LPS, B. melitensis, and B. abortus CβG induced a robustexpression of CD80, CD86, CD40 and MHC class II molecules, although inslightly lower levels, were in cells incubated with B. abortus CβG. Bycontrast, Ralstonia CαG and MβCD failed to significantly activate DC.

Analysis of cytokines secretion corroborates surface marker expression.B. melitensis and B. abortus CβG induced TNF-alpha and IL-12 secretion.In response to Ralstonia CαG, DCs hardly produced TNF-alpha and IL-12and in response to MβCD none of the cytokines were secreted.

The above data first demonstrated that cyclic glucan-dependent DCactivation greatly depends on the molecule structure and secondly thatcholesterol extraction does not have any effect on immune systemresponses in DC.

DC-Activation By CβG Requires TLR4, MyD88 And TRIF, But Is Independentof CD14.

We analysed the contribution of TLRs and adaptors for the recognition ofCβG. MyD88 and TRIF are adaptor molecules involved in TLR signalling.MyD88 is involved in the TLR1, 2, 4, 5, 7, 8, 9, IL-1R and IL-18Rpathways whereas TRIF is unique to TLR3 and TLR4. BMDC derived fromTLR4, TLR2, MyD88, TRIF, TRIF/MyD88 and CD14 KO mice were treated withCβG. No activation could be observed in TLR4, Myd88, Myd88/TRIF and TRIFKO mice-derived BMDC stimulated either by E. coli LPS and B. melitensisCβG, as indicated by measuring surface expression of co-stimulatorymolecules and secretion of pro-inflammatory cytokines such as IL-12,IL-6 and TNF-α (FIG. 2 And B). These results show that B. melitensis CβGinduce DC maturation via the activation of the TLR4 pathway. Todelineate the pathway involved in DC maturation induced by B.melitensis, CβG activation was tested in BMDC derived from MyD88KO,TRIFKO and double MyD88/TRIFKO mice. Similarly to E. coli LPS, theinduction of IL-12 by B. melitensis CβG was hampered in BMDC derivedfrom MyD88-KO and TRIF-KO mice, No IL-12 secretion was observed indouble MyD88/TRIF KO when cells were incubated with Brucella CβG incontrast to DC treated with curdlan, a linear α-1,3 glucan fromAlcaligenes faecalis. Curdlan is known to interact with the key β-glucanreceptor Dectin-1 in a MyD88/TRIF-independent manner.

LPS recognition involves the LPS-binding protein (LBP), CD14 and theTLR4/MD2 complex. Secretion of pro-inflammatory cytokine such as IL-12were completely abolished in E. coli LPS-treated CD14 KO BMDCs as wellas cell surface expression of co-stimulatory molecules, MHC-II. Incontrast, activation of CβG-treated BMDCs was independent of CD14. Thefact that CβG does not require CD14 to activate the TLR4 pathwayhighlights a different mechanism of recognition than LPS. Altogether,these results demonstrate that CβG is a novel TLR4 agonist.

Potent biological inducers such as LPS display high toxicity andimmunogenicity, properties that hamper the use of these molecules asadjuvant. It was therefore relevant to explore these properties in thepurified Brucella CβG preparations. In contrast to E. coli LPS, BrucellaCβG was not toxic for cell cultures or animals (Table 1) and did notinduce the generation of antibodies in mice, rabbits or in naturallyinfected bovines (not shown). These results corroborate previous resultsshowing that in cell culture, CβG was not toxic even at very highconcentrations (10 mM). Thus, CβG is a TLR4 agonist that does notdisplay the toxic properties of LPS.

Brucella CβG Induce A CD8 Response In Vivo.

Inventors investigated the ability of cyclic glucan to induce a CD8+ Tcell response to an exogenous free antigen. Inventors transferred CD8+Ly5.2 CFSE+ OT-I T cells into C57B1/6 Ly5.1 mice which were thenimmunized (s.c.) either with PBS alone (group 1), OVA alone (group 2),OVA with cyclic glucan (group 3) or OVA with known adjuvants such asPoly I/C (group 4) or IFA (group 5). Three days after the immunizationinventors could observe an OT-I T cell proliferation in the draininglymph node in mice of all groups except the control immunized with PBS.We next explored the surface expression of activation markers inCD8+CD45.2+ population. Inventors analyzed by flow cytometry theup-regulation of CD25, CD44 expression and the down-regulation of CD62L,which correlates with T cells migration from lymph nodes to sites ofinfection. There was no cellular activation in mice vaccinated with PBSalone or PBS with OVA. However in the group immunized with OVA andcyclic glucan, inventors could observe a CD25 and CD44 up-regulation, aswell as a strong down-regulation of CD62L expression, which is higherthan in groups immunized with known adjuvants as Poly:IC.

These data indicate CβG can induce CD8+T cell proliferation andactivation in vivo.

Brucella CβG Induce Maturation of Human DCs.

Inventors investigated whether B. melitensis CβG was also a stimulus forhuman monocyte-derived DC. Human DCs were differentiated from monocytesobtained from human peripheral blood in the presence of GM-CSF and IL-4for 6 days or GM-CSF and IFN for 3 days. Inventors therefore tested theeffect of various concentrations of B. melitensis CβG on DCs maturation.In IFN-differentiated DC, the up-regulation of CD80, CD83, CD40, HLA-DR,HLA-ABC and CD86 on the cell surface was detected in a dose dependentmanner after treatment during 24 h with B. melitensis CβG (FIG. 2A). Asin mouse DC, the lower concentration used (0.2 μM) was not able toinduce this DC phenotype. The same result was present forIL-4-differentiated human DCs. Inventors further demonstrated that CβGefficiently activate human blood mDCs, resulting in significant amountsof IL-1b, TNFα, and IL-12, as observed in mouse DCs. In particular, CβGwas more potent than E. coli LPS for the induction of IL-1b from bloodmDCs (FIG. 2B).

mDCs activation with CβG resulted in increased allogeneic naïve CD4+ Tcell proliferation. It also resulted in enhanced Flu M1-specific CD8+ Tcell responses as well as MART-1specific CD8+ T cell responses. AlthoughCβG was more efficient than E. coli LPS for CD4+ T cell proliferation,both CβG and E. coli LPS resulted in similar levels of Flu M1-specificCD8+ T cell responses as shown in the cross-presentation assay.

Taking together, these results indicated that Brucella CβG is apan-activator of DC and macrophages.

Brucella CβG Is Not Endotoxic In Vivo And In Vitro.

Because CβG was able of triggering a strong activation and because itseemed that the mediated activation was dependent on the TLR4 pathway,inventors looked at the possible endotoxicity generated in vitro and invivo. Different assays were used, in particular the Limulus amoebocytelysate assay, the DL50 in Swiss mice injected by the various agonistsand the toxicity in macrophages as measured by Chromium release. Table Ishows that CβG was not toxic at all compared to E. coli LPS and BrucellaLPS. As expected, compared to Brucella LPS, E. coli LPS was able toinduce an endotoxic shock at very low concentration.

TABLE I B. melitensis CβG is devoid of endotoxic properties Limulusamoebocyte LD50 Toxicity in lysate in mice macrophages E. coli LPS 0.1ng/ml  65 μg  25 μg B. melitensis LPS 0.1 ng/ml >250 μg  >50 μg B.melitensis CβG >105 ng/ml   >500 μg >100 μg

Limulus amoebocyte lysate from Pyrogent MA Bio products Inc.Walkersville USA. Method was performed according to the manualinstruction.

LD50 was determined in Swiss mice (20 to 22 g), with 0.1 ml of theappropriate LPS concentrations. Deaths were recorded at 12, 24, 48 and72 h, and the 50% lethal dose was determined by the probit method. Theprobit Method. (20). Toxicity for peritoneal macrophages was performedusing the Chromium release method.

EXAMPLE 2 Adjuvant Activity of Brucella CβG Methods

The adjuvant activity of CβG for induction of antibodies against snakevenom was evaluated according to the method proposed by (24). Briefly,the venom of Bothrops asper diluted in PBS was mixed with calciumalginate or with PBS (control) and the mixture was emulsified. Then CD-1mice were injected subcutaneously with the mixture, to give anequivalent to 20 μg of venom in a volume of 50 μl/mouse. Another groupof mice was injected through the same route with the same mixture ofcalcium alginate and venom but also including 100 μg of B. abortus CβGper mouse. Finally a group of mice was only inject venom diluted in PBS.Immunized mice were bleed from the day “0” to day 30, and the antibodyagainst the venom of B. asper were determined by indirect ELISA usingplates coated with whole venom antigen and as conjugate anti-mouseIg-horseradish peroxidase (24). The independent values of the differentgroups of mice were evaluated and the differences assessed byunidirectional

ANOVA analysis, followed by Fisher significance (PLSD) analysis.

Results

Calcium alginate is the immunoadjuvant classically used to induce animmunological response toward venom. As shown in FIG. 4 CβG synergizeswith calcium alginate for the production of anti-venom immunoglobulinsafter 21 and 28 days (FIG. 4A). The kinetic of antibodies production isthus accelerated and the adjuvant activity is higher than the oneobserved with calcium alginate alone (FIG. 4B).

REFERENCES

Throughout this application, various references describe the state ofthe art to which this invention pertains. The disclosures of thesereferences are hereby incorporated by reference into the presentdisclosure.

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The invention claimed is:
 1. A method of enhancing an immune responseagainst one or more isolated or purified antigens in a mammalian subjectin need thereof comprising administering to the subject concomitantlywith a pharmacologically effective amount of said one or more isolatedor purified antigens and an adjuvant effective amount of animmunoadjuvant comprising at least one purified, isolated or synthesizedcyclic beta glucan (CβG), wherein said at least one CβG stimulates thesubject's immune system and induces dendritic cell activation andmaturation in said subject, thereby enhancing said subject's immuneresponse to said one or more isolated or purified antigens.
 2. Themethod of claim 1 wherein said at least one CβG is substituted with atleast one natural substituent residue.
 3. The method of claim 2 whereinthe at least one natural substituent residue is a succinyl residue. 4.The method of claim 2, wherein the at least one natural substituentresidue includes a number of natural substituent residues which rangebetween 0 to
 25. 5. The method of claim 1, wherein the at least one CβGis a CβG obtained from B. melitensis and/or Brucella abortus.
 6. Themethod of claim 4, wherein the number of natural substituent residuesranges between 0 to
 3. 7. The method of claim 1, wherein said immuneresponse includes both an adaptive immune response and an innate immuneresponse.
 8. The method of claim 1, wherein said immune responseincludes pro-inflammatory cytokine secretion and activation andproliferation of both CD4 and CD8 T cells.